Portable water quality instrument

ABSTRACT

A hand-held microfluidic testing device is provided that includes a housing having a cartridge receiving port, a cartridge for input to the cartridge receiving port having a sample input and a channel, where the channel includes a mixture of Raman-scattering nanoparticles and a calibration solution, where the calibration solution includes chemical compounds capable of interacting with a sample under test input to the cartridge and the Raman-scattering nanoparticles, and an optical detection system in the housing, where the optical detection system is capable of providing an illuminated electric field, where the illuminating electric field is capable of being used for Raman spectroscopy with the Raman-scattering nanoparticles and the calibration solution to analyze the sample under test input to the cartridge.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/280,651, filed Feb. 20, 2019, abandoned which is a continuation ofU.S. patent application Ser. No. 14/198,163, filed Mar. 5, 2014 now U.S.Pat. No. 10,254,229. U.S. patent application Ser. Nos. 16/280,651 and14/198,163 are incorporated by reference.

STATEMENT OF GOVERNMENT SPONSORED SUPPORT

This invention was made with Government support under contactIIP-1058590 awarded by National Science Foundation. The Government hascertain rights in this invention.

FIELD OF THE INVENTION

The invention relates to fluid analysis. In particular, the inventionrelates to a hand-held microfluidic analysis device that can be used inremote applications.

Chemical mixture separation is important to many fields. While a varietyof approaches exist for chemical separations, chromatography andelectrophoresis are two of the most commonly used analytical methods.

Chromatography is a set of laboratory techniques used to separateconstituents from a chemical mixture. Chromatography is used ineverything from water and food safety to biotechnology and drugdiscovery. It is also a common technique used in standard laboratoryprocedures and cutting-edge scientific research, where liquidchromatography, size-exclusion chromatography, affinity chromatographyand high-pressure liquid chromatography are traditionally used.

In liquid chromatography, the unknown sample is dissolved in a liquidmobile phase, which is then over a stationary phase. The analyte ofinterest remains with, or is slowed by, the stationary phase, separatingit from the overall mobile phase mixture.

Liquid chromatography can be further divided by the stationary phase,comprising three methods: ion-exchange, size-exclusion, and affinitychromatography. In ion-exchange chromatography, the stationary phasecontains charged functional groups, which interact with the charge onthe analyte. This charge will affect the migration time through thechromatography system, separating the analyte from the overall sample.

Size-exclusion chromatography, or gel permeation chromatography,separates constituents based on size, passing the mobile phase through aporous medium that only passes particles below a certain size.

Affinity chromatography is based on selective covalent bonding with themobile phase. For example, proteins or polymers tagged with a specificlinker can be isolated with the appropriate linker analogue.

General improvements in liquid chromatography have increased theefficiency and resolution, leading to the more common description ofhigh-pressure (or performance) liquid chromatography (HPLC). Suchseparations typically require macroscopic volumes of material—the mobilephase may be a few milliliters or more. Additionally, these separationscan take hours to process. And, even with these drawbacks, the resultsare still rather crude single-monomer polymer separation across a broadstripe of analytes is a significant challenge.

An alternative to HPLC is electrophoresis, where charged molecules areseparated in an electric field. FIG. 1 shows a prior art schematicillustration of electrokinetic flows 100. The electroosmotic 102 andelectrophoretic 104 flows of particles 106 scale linearly with theelectric field E by a mobility factor μ inside a channel 108, where theelectric field is created by a voltage supply 110 across the length ofthe channel 108. The arrows are shown in opposite directions forillustration, where they may point in either direction depending oncharges and material properties. This separation of the components of amixture of charged molecules is an important scientific andtechnological process, including analytical methods such as DNAsequencing and preparative methods such as the purification of proteins.Successful separation of a mixture of polyelectrolytes by an appliedelectric field according to charge or mass depends on symmetry-breakingmechanisms between the driving force, related to the electric field, andthe friction offered by the medium, such as a buffer solution with orwithout a matrix such as a gel. Accordingly, if the driving force andthe friction force scale the same way with charge or length, the ratioof these quantities is then independent of charge or length, andseparation is not achieved. There are many ways to achievesymmetry-breaking for polyelectrolytes such as DNA, ranging from the useof gel matrices for sequencing relatively short DNA fragments in aconstant electric field, to pulsed-field gel electrophoresis forseparating large DNA fragments, or to creating asymmetric molecules forseparation in free solution.

There are multiple approaches to electrophoresis. Capillaryelectrophoresis separates components within a glass capillary. Here, theproperties of microfluidic flow within such a capillary improveefficiency and reduce separation times. Known instruments can reduce theseparation time to 30 minutes, while allowing the use of a fewmicroliters of material. For fields where materials are expensive andrare, such as drug discovery, capillaries offer tremendous benefits.

Furthermore, in the last few years, microfabricated capillaryelectrophoresis devices have entered the market. These devices offerparallel processing with a few to dozens of simultaneous separations.Microfabricated devices also work with smaller sample sizes. Theseadvantages are both increasingly important in biotechnology, as manysamples are of limited material quantities. Commercial microfabricatedcapillary electrophoresis systems are being used for DNA sequencing, RNAanalysis, protein separations, and even cell content studies.

The ability to separate a chemical mixture into constituents isabsolutely necessary for all of analytical chemistry. Improvements inspeed, quality, efficiency, or resolution of separation techniques arenecessary enhance the behind-the-scenes laboratory work that ensures thequality of everyday products. These products can be categorized in thefields of pharmaceuticals, laboratory, environmental, food/beverage, andacademic. Each of these segments has a broad impact across all ofsociety, such as quality-control analysis, where imported food productsmust be analyzed for hazardous materials, or water and soil must bemeasured for pollutants before entering public consumption.

Many liquid chromatography methods are used in environmental analysis,such as water and soil quality analyses, where measuring organiccompounds or mineral-content levels in water is handled by experiencedlab technicians operating chromatography tools. These processes areexpensive and time-consuming. Nevertheless, the application ofchromatography in these fields is tremendous and pervasive. Thus, thebroader impact of new techniques to speed and improve chemicalseparations is wide-ranging and important.

While recent electrokinetic separations have improved separation speedand resolution for charged molecules over traditional chromatographictechniques, such improvements are still lacking for many particlesincluding charged and uncharged molecules.

Capillary electrophoresis provides improvements in speed and resolutionover LC. Furthermore, capillary electrophoresis works effectively inparallel systems and with microscopic volumes. When molecules areuncharged, electrophoretic methods have been ineffective, the optionsfor separating such molecules are limited to older LC techniques, suchas size-exclusion or affinity chromatography.

Uncharged polymers are important in many everyday products. For example,poly(ethylene glycol) is used in a multitude of medical applications: inlaxatives, in skin creams and eye drops, and for delayed protein drugdelivery. The polymer poly(vinyl alcohol) is used extensively inproducts ranging from children's putty to adhesives. Furthermore, underelectrophoretic conditions, free-draining coils, such as DNA, areeffectively uncharged as their drag-to-charge ratio is uniform, whereDNA will not separate in an electric field without a symmetry-breakingmechanism. Additionally, many proteins and peptides are effectivelyuncharged; electrophoretic separation of these important biomaterials isnot possible with additional processing steps. As uncharged polymers arenecessary components of everyday materials, improved separations ofthese materials will improve the safety and quality of these products.

Accordingly, there is a need to develop hand-held low-cost microfluidicseparation device separating charged and uncharged particles, where themethod has broad applications in environmental, biotechnological, andchemical processing. A further need exists for such a device thatprovides detection resolution at the part-per-billion (ppb) level.

SUMMARY OF THE INVENTION

To address the needs in the art, a hand-held microfluidic testing deviceis provided that includes a housing having a cartridge receiving port, acartridge for input to the cartridge receiving port having a sampleinput and a channel, where the channel includes a mixture ofRaman-scattering nanoparticles and a calibration solution, where thecalibration solution includes chemical compounds capable of interactingwith a sample under test input to the cartridge and the Raman-scatteringnanoparticles, and an optical detection system in the housing, where theoptical detection system is capable of providing an illuminated electricfield, where the illuminating electric field is capable of being usedfor Raman spectroscopy with the Raman-scattering nanoparticles and thecalibration solution to analyze the sample under test input to thecartridge.

In one aspect of the invention, the chemical compounds can includethiols, amines, silanes, polymeric particles, metallic particles, crownesters, cysteamine, cystamine, diethylaminethanethiol, mercaptopropionicacid, 1-propanethiol, octanethiol, octyldecanethiol, polystyrene, iron,or silica.

According to another aspect of the invention, the calibration solutioncomprises isotopes of the sample under test.

In a further aspect of the invention, the calibration solution comprisesa chemical composition in the sample under test.

BRIEF DESCRIPTION OF THE DRAWINGS

The objectives and advantages of the present invention will beunderstood by reading the following detailed description in conjunctionwith the drawing, in which:

FIG. 1 shows a prior art schematic illustration of electrokinetic flows.

FIG. 2 shows an electric double layer leading to electroosmotic flowaccording to the present invention.

FIG. 3A shows a non-uniform flow channel geometry.

FIG. 3B shows x-velocity results from a finite element analysis of theflow channel of FIG. 3A.

FIG. 3C shows y-velocity results from a finite element analysis of theflow channel of FIG. 3A.

FIG. 4 shows numerical results on total polymer displacement ofdifferent channel geometries according to the present invention.

FIG. 5 shows a schematic of a device having a long region of undularychannels, coupled to inlet and outlet channels according to the presentinvention.

FIG. 6A shows a first step in a process for fabricating a microfluidchannel device.

FIG. 6B shows a second step in the process of FIG. 6A for fabricating amicrofluid channel device.

FIG. 6C shows a third step in a process of FIG. 6A for fabricating amicrofluid channel device.

FIG. 6D shows a fourth step in a process of FIG. 6A for fabricating amicrofluid channel device.

FIG. 7 shows a schematic drawing of a polymer separation apparatusaccording to the present invention.

FIG. 8 shows a top view of one embodiment of the microfluidic mixingdevice according to the present invention.

FIG. 9 shows a side cutaway view of a detection region of themicrofluidic device in FIG. 1 according to the present invention.

FIG. 10 shows a top view of a detection region with a liquid sieve atone end according to the present invention.

FIG. 11A shows flexible microfluidic walls in an unconstricted state.

FIG. 11B shows the flexible microfluidic walls of FIG. 11A in aconstricted state.

FIG. 12A shows a top view of an alternative form of nanoparticledelivery device.

FIG. 12B shows a cross section of the nanoparticle delivery device ofFIG. 12A.

FIG. 12C shows another view of the cross section of FIG. 12B with anapplied electric potential.

FIG. 13A shows a front view of an analysis instrument, illustrating ascreen to present information to the user and buttons for user input.

FIG. 13B shows a bottom view of the analysis instrument of FIG. 13A,illustrating a slot for inserting an analysis cartridge.

FIG. 14 shows an example optical system contained within the analysisinstrument according to the present invention.

FIG. 15A shows a block diagram presenting subcomponents of a system.

FIG. 15B shows a flow chart of the use of the system of FIG. 15A.

FIG. 16 shows an example of data presented to the user according to thepresent invention.

FIG. 17A shows a top view of an analysis cartridge.

FIG. 17B shows and a side view of the analysis cartridge of FIG. 17Aillustrating a shell, chip base, chip lid, sealing gasket, andprotective film of the analysis cartridge.

FIG. 18 shows a hand-held microfluidic testing device having a housingand cartridge, where the cartridge shows a calibration solution in thechannel, according to one embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Although the following detailed description contains many specifics forthe purposes of illustration, anyone of ordinary skill in the art willreadily appreciate that many variations and alterations to the followingexemplary details are within the scope of the invention. Accordingly,the following preferred embodiment of the invention is set forth withoutany loss of generality to, and without imposing limitations upon, theclaimed invention.

A new technique for separating uncharged and charged particles isprovided, where the method has broad applications in environmental,biotechnological, and chemical processing. An electrokinetic approach isprovided for the separation of uncharged polymers. Typically, unchargedmolecules, such as certain peptides, proteins, and commerciallyimportant polymers, are not influenced by an electric field, althoughthey will interact with a moving buffer solution. When this movingbuffer solution has a spatially uniform velocity profile, the unchargedmolecules will not separate by length or size. Conversely, when themoving buffer solution has a non-uniform velocity profile, molecules canbe separated based on size.

While an electric field does not affect uncharged molecules directly, itdoes give rise to a bulk flow of a buffer solution. This bulk fluidmovement past a stationary solid surface, called electroosmosis, is dueto the formation of a charged double layer at the solid-liquidinterface. FIG. 2 shows a schematic drawing of an electric double layerproviding electroosmotic flow 200. As shown a first charge layer 202 isfixed to channel wall surface 204, while the opposite charges 206 remainmobile in solution 208. At many liquid-solid interfaces, dissociatedliquid ions, such as water ions, will interact with the solid surface,creating a charged double layer 210, where an applied electric field(not shown) is able to move the mobile ions 206 within the double layer210, dragging the bulk solution 208 and any solvated molecules (notshown) along the microchannel 212. The solution can be water, phosphatebuffered saline, TTE (0.5 M Tris, 0.5 M TAPS, 0.02 M EDTA) or anywater-based solution.

According to the current invention, non-uniform electroosmotic flowprovides separation of uncharged molecules in free solution. The currentinvention uses the properties of Brownian motion and how it affects therandom motion of small molecules more than large molecules.Specifically, small molecules exhibit a higher probability of spendingtime away from the center of a channel having non-uniform electroosmoticflow than the large molecules, resulting in a longer average path lengthfor the small molecules and contributing to a lower effective mobility.The current invention provides a method of enabling large molecules toexit the channel ahead of the small molecules, where the small moleculeshave a longer travel time through the channel.

As stated, a non-uniform electroosmotic flow is generated by varying thechannel geometry along the x-axis, resulting in a distribution ofvelocities in the x-direction along the y-axis. As a particle movesalong the y-axis, away from the center of the channel, the curved flowlines of the channel walls result in longer path lengths. Because thesmaller molecules have a greater propensity to diffuse laterally,size-dependent separation occurs, where smaller molecules fall behindthe larger molecules within the solution as they migrate along thecolumn length. Conversely, the larger molecules tend to spend more timealong the center of the channel and progress through the column at afaster rate.

FIGS. 3A-3C show channel geometry and flow diagrams 300 according to oneembodiment of the invention. FIG. 3A shows a non-uniform channel 302having a narrow span 304 and a wide span 306, where a geometry parametera is defined as the ratio of the widest 306 to narrowest 304 regions.Shown in FIGS. 3B and 3C are x-velocity and y-velocity results,respectively, from a finite element model. Here, Navier-Stokes andmomentum conservation equations are used, and a force term is added dueto the applied electric field, where ρ is the fluid density, ν is thevelocity, μ is the viscosity, κ is the Debye length, ρ is the pressure,ε is the dielectric constant, ψ is the potential due to the electricdouble layer, and φ is the applied potential, giving:

${{\rho\left\lfloor {\frac{\partial v}{\partial t} + {\left( {v \cdot \nabla} \right)v}} \right\rfloor} = {{- {\nabla p}} + {\mu{\nabla^{2}v}} + {\varepsilon\varepsilon_{0}K^{2}\psi{\nabla\varphi}}}}{{\nabla \cdot v} = 0}$

The total electric potential in the microfluid channel can be describedas the sum of the electric double layer potential and the appliedpotential. The Debye-Hückel approximation is used to simplify theexpression for ψ, allowing both electric potentials to be determined byPoisson's equation.

A finite element analysis software package was used with standard valuesfor the density, dielectric constant, and viscosity of water, plus theDebye layer parameters ξ=−40 mV and κ=1.13 μm⁻¹. The solution wasdiscretized on a 400×400 grid, and introduced into the polymer flowsolver.

To demonstrate the efficacy of the current invention, the flow patternwas generated on four geometries. The flow for α=1.0, 1.9, 3.0, 4.1, and7.0 were modeled, with each respective geometry repeated periodically.For each geometry parameter, the velocity was scaled at the channelcenter to the value for α=4.1. A parameter of unity corresponds to aconstant flow rate across and along the entire channel, thuscorresponding to a uniform electroosmotic flow.

The polymer flow was modeled using an exemplary discrete, worm-likechain model. The model consists of N beads of radius a connected by N−1springs. For N beads, with positions r_(i), the equations of motion aregiven by:

${\overset{.}{\overset{\_}{r}}}_{j} = {{\sum_{\,{j = 1}}^{\, N}{{\overset{¯}{D}}_{ij}\overset{¯}{F_{j}}}} + {{{\overset{¯}{N}}_{i}(t)}.}}$

This equation was solved directly by a second-order Runge-Kutta methodfor stochastic differential equations. All motion in the z-direction wasset to zero. The force acting on each bead, F_(j), has three components:the Stokes' force from the moving fluid with velocity v_(j) at the beadposition, and the inter-bead spring and bending potentials, as providedin Volkel and Noolandi.

The fluid velocity was taken from the finite element model, with linearinterpolation from the output grid to the particle coordinates. Theconstants from these equations are: bond length, b=2 a; persistencelength, P=5 b; and spring constant, h=100 b. The terms Ni(t) areGaussian-distributed random numbers with zero mean and variance.

N_(i)(t)N_(j)(t′)

=2κ_(b)TD_(ij)δ(t−t′).

The Ni terms were calculated at each time-step using a Choleskydecomposition. The self-diffusion terms of the diffusion tensor aregiven by Volkel and Noolandi. For bead-bead hydrodynamic interactions,the terms are given by the Rotne-Prager approximation. For eachgeometric parameter α, the strings were modeled at each of the lengthsL=5, 10, 15, 20, 25, 30, and 40 beads.

The first observation is the final position of the polymers as afunction of length and geometry. Uniform flow (i.e., α=1) results inlonger polymers traveling slightly less distance than shorter polymers.In contrast, as the geometry parameter increases, the longer polymerstravel farther, with increasing separation based on length.

The position along the channel of representative polymers is illustratedin FIG. 4 that shows numerical results on total polymer displacementaccording to the current invention. For each geometry parameter, thesymbol indicates the final position of the 5-bead string. While eachgeometry parameter results in the polymers moving different distances,the polymers end in regions with approximately the same velocity in thex-direction. Three representative flow lines are shown for α=4.1.

Note, in contrast to a uniform flow field, longer polymers travelfurther than shorter polymers, and as the geometry parameter increases,the travel distance between short and long polymers increases.

Additional data illustrating the effects of polymer length ondisplacement is presented in FIG. 4 . For the largest geometryparameter, the difference in distance between a 5-bead string and a40-bead string is nearly 5%. This difference contrasts with the uniformflow situation, where the distance difference is close to zero.

The current invention provides a novel and powerful technique for theseparation of charged and uncharged polymers. Capillary electrophoresisenables faster, more accurate and smaller sample size analysis overhigh-pressure liquid chromatography, but it only works with chargedmolecules. The current invention, unlike capillary electrophoresis,provides analysis of both charged and uncharged molecules.

Separation of uncharged polymers, where longer uncharged polymers willtravel further along a channel than short uncharged polymers innon-uniform electroosmotic flow, enables polymer discrimination tosingle monomer resolution, according to the current invention.Single-monomer resolution separation of a poly(ethylene glycol) mixtureis of critical importance to commercialization; if the device cannotseparate the polymers with high resolution, the usefulness decreases.

FIG. 5 shows a schematic of a device 500 having a long region ofundulary channels 502, coupled to at least one inlet channel 504 and atleast one outlet channel 506, according to the present invention. Sampleinsertion channels 508 are shown perpendicular to the inlet channel 504.A method of using electrokinetics for separating particles in a buffersolution is provided by the current invention. The column 502 has anon-uniform internal longitudinal cross-section, and the column can havea shape that can be linear, curved, circular, or spiral. At least onemain inlet 504 and at least one main outlet 506 are provided, where thesolution is input to the main inlet 504 and output from the main outlet506. At least one sample inlet 508 and at least one sample outlet 510are provided, where the particle (not shown) is introduced to the column502 from the sample inlet 508 and fractionated samples are eluted fromthe sample outlet 510, whereby quality control and further analysis areenabled. An electric field is applied to the solution (see FIG. 7 ) inthe column to generate a charged double layer (see FIG. 2 ) at asolid-liquid interface within the column, where the electric filed movesions within the double layer, and a non-uniform velocity profile (seeFIG. 3 ) is induced to the buffer solution, where the moving ions carrythe particles along the column and the particles are separated accordingto size or charge.

According to one embodiment of the invention, the non-uniform channel502 internal longitudinal cross-section has a generally counterundulating-shape profile, where the counter undulation is between afirst wall cross-section and a second wall cross-section. The undulationcan have a peak to peak distance in a range from 1 μm to 500 μm.Further, the undulation can have an undulation linear density rangingfrom 0.05 peaks/μm to 1 peak/μm. Further, the undulation first wallcross-section and the second wall cross-section have a ratio, or valueof α with a widest separation and a narrowest separation that is greaterthan or equal to one.

In one exemplary aspect of the invention, the devices may be fabricatedfrom glass wafers, such as Corning 7740. The surface chemistry ofmicrofabricated glass devices is similar to capillaries, allowing theuse of the same experimental techniques as used in capillaryelectrophoresis work. Other materials for electrophoresis can includemolded plastic parts or other transparent wafers, such as quartz.

FIGS. 6A-6D show a process flow for fabricating a microfluid channeldevice 600 according to the present invention. In FIG. 6A the processstarts by providing a glass wafer 602 and applying a lithographicpattern photoresist 604 (see FIG. 6B) on the glass wafer 602. The wafers602 are etched in a buffered oxide etch (see FIG. 6C), where the etchedregions provide microfluidics channels 606. This etch is isotropic,creating half-cylindrical channels 606. A dry etch would result in arectangular cross-section, if that were so required, according to oneaspect of the invention. Access holes 608 are provided in a secondglass-capping wafer 610 (see FIG. 6D). Once the wafers 602 are etched,the capping wafer 610 is aligned and thermally bonded thereto, accordingto one aspect of the invention. Unlike capillary electrophoresis, thecurrent invention does not suppress electroosmosis, so internal channelcoatings are unnecessary.

FIG. 7 shows a schematic drawing of a polymer separation apparatus 700according to one embodiment of the current invention. A high-voltagepower supply 702 is used to drive the polymers (not shown) along thechannel 704, while a fluorescence microscope 706 records thefluorescently-labeled polymers as they pass. It should be understoodthat there are numerous possible methods to identify and record thepolymers within the apparatus, some examples include fluorescence, Ramanspectroscopy, amperometry, color, or mass spectrometry which areembodied in the current invention.

As an example operation, a polymer containing solution is introduced tothe main undulary channel 704 (see FIG. 6 for example) from the sideinjection channel 708. A voltage is applied along the main channel 704,allowing the polymers to travel down the channel 704. A change in chargemay affect the flow rate, which can effect the retention time. Bynormalizing to the flow rate, a normalized retention time becomes thesame for each run. This information enables quantitative measurement ofpolymer length based on transit time, according to one aspect of thecurrent invention. The particles can have a particle size ranging from 1nm to 500 μm.

The current invention provides a microfluidic separation device that isuseful for surface-enhanced Raman spectroscopy (SERS) and otherdetection methods. Raman spectroscopy in general provides a chemicalsignature for a compound, but the Raman signal is generally too weak forpart-per-billion detection levels. However, when a metallic nanoparticlethat is smaller than the wavelength of light is introduced into thesample, the illuminating electric field will create surface plasmonresonances if there are free electrons in the nanoparticle, where thenanoparticle can be gold, silver, or copper beads, for example. Theseoscillating charges create an enhanced local electric field alongcertain directions. This field results in a much stronger Ramanresponse. SERS experiments are often characterized by “hot spot”regions. Here the SERS signal reaches single-molecule detectioncapabilities. These regions are most likely due to nanoparticle toalignments that create even larger electric field enhancements.

Using SERS for analyte detection has been under study. It is believedthe large signal enhancement creates new opportunities to measure verysmall concentrations: picomolar, femtomolar, and potentially even singlemolecules. The challenge with SERS is creating an interaction betweenthe analyte and the metal surface. The highest-sensitivity studies relyupon binding events to bring the molecules into close contact. Whilevery sensitive, this approach is limited to measuring a previouslydecided set of analytes for which the nanoparticles are prepared. Thebinding does not need to be specific; for example, treatments withoctadecylthiol have been used successfully for SERS on planarsubstrates.

According to one aspect or the current invention, a sensitive detectionsystem incorporated in a portable device is provided. The inventionincludes packing sections along a microfluidic separation channel withnanoparticles, for example gold nanoparticles, at a high density. Theinvention creates many hot spots simply through particle density. Theinvention uses microfluidic delivery and narrow channel geometries totrap signal-enhancing particles at a detection location within a longerseparation channel. Referring now to the figures, FIG. 8 shows a topview schematic of a microfluidic separation device 800 having a mainchannel 802 spanning from a fluid input 804 to a fluid output 806. Themicrofluid separation device 800 includes at least one sample loadingport 808 connected to the main channel 802 by a sample loading tochannel 810. Separation regions 812 are disposed down stream from thesample loading channel 810. The invention further includes at least onedetection particle loading port 814 connected to the main channel 802 bya detection particle channel 816. At least one detection region 818 isdisposed down stream from the detection particle channel 816. As shownin the exemplary device of FIG. 8 , the main channel 802 is intersectedby two perpendicular sample loading channels 810 to load the sampleunder study into the main channel 802, while the detection particlechannel 816 is for loading the nanoparticle markers into the mainchannel 802.

FIG. 9 shows a side view of a detection region 818 of the microfluidicmixing device 800 in FIG. 8 according to the present invention. Proximalto the intersection of the detection particle channels 816 and the mainchannel 802 are geometric constrictions 900 that trap the nanoparticles902 within the detection region 818. Further shown are arrows toindicate the flow direction of the fluid within the microfluidseparation device 800. The fluid moves the nanoparticles 902 along theflow path to compact them within the detection region 818. The currentinvention relies purely on proximity by creating a region densely packednanoparticles 902. According to one exemplary structure of theinvention, tight packing density (close-packed spacing) predicts amaximum volume ratio of 74% spherical nanoparticles for a microfluidicchannel region 818 that is 50 μm in length and has a 25 μm width anddepth, loaded with 40-nm gold nanoparticles 902, this type of volumepacking will have a surface area nearly 700 times greater than thesurface area of the channel region. Furthermore, the narrow regionsbetween nanoparticles 902 with the non-linear path through the matrixwill increase interactions. The current invention provides a sensitivityrequirement of detecting materials in parts per billion.

In the base configuration of the current invention, included is a mainchannel 802 with at least one crossing sample loading channel 810 and atleast one nanoparticle loading channel 816, and the detection region818. The detection region 818 has geometric constraints 900 that preventparticles 902 of a certain size from entering the main channel 802 ineither direction, or from continuing past the detection channel 818. Thenanoparticles 902 may be metallic, such as gold, copper, silver,fluorescent particles, magnetic particles, particles having bindingchemistry, latex particle, polystyrene particles or quantum dots forsurface-enhanced Raman scattering. According to one aspect, theparticles are on the order of 10 nm to 10 μm. The particles 902 may alsobe fluorescent beads designed to bind with an analyte of interest for anELISA-type signaling approach. These particles can be loaded using anytype of fluid driving mechanism such as electroosmosis, electrophoresis,fluid pressure, moveable wall pressure, undulary electroosmosis,undulary electrophoresis, undulary fluid pressure or undulary moveablewall pressure. Note that between the sample input channel 810 anddetection region 818 can be a separation region 812 that isolatesindividual compounds (undulary electroosmosis, electrophoresis, orchromatography) before entering the detection region 818.

It should be apparent there are many geometries may be used to createthese detection regions 818. The constrictions 900 can occur in thevertical direction, reducing the size of the detection region 818 fromtop to bottom. This approach requires etching short depths orsacrificial layers. The constrictions 900 can also occur in thehorizontal direction, which would rely upon lithographic abilities todefine the narrowest gaps.

According to one aspect of the invention, the Raman signal can befurther increased by using chemistries, both non-specific and specific,to bind analytes to the nanoparticles 902. With over a billionnanoparticles 902 in each detection region 818, along with multipledetection regions 818, a separation column 802 could hold a large numberof modified nanoparticles 902. For example, with five detection regions818, each holding two hundred different bindings, this system 800 coulddetect one thousand compounds while maintaining greater than fivemillion nanoparticles 902 per region.

FIG. 10 shows an alternate embodiment 1000 of the invention, where thedetection region 818 includes a sieve material 1002 that allows thecarrying fluid to continue moving but stops the detection particles 902.For example, a molecular sieve will allow water to pass under pressurethrough atomic level openings in the material, but will block passage oflarger particles.

FIGS. 11A and 11B show another embodiment of the invention 1100 thatincludes a reconfigurable detection region. According to the currentembodiment 1100 the main channel 802 can be constructed from a flexiblematerial, such as silicone elastomers. If a bladder region 1102 wasplaced in near proximity to the main channel 802, any pressure appliedto the bladder 1102 will expand into the main channel 802, and provide aconstricting region 900 to the channel 802. This approach allows fordetection regions 818 to be repeatedly created and released, thusallowing for repeated use with different detection particles. It alsoallows one generic design to use particles 902 of different sizes, asthe channel can be configured for any size constriction.

FIGS. 12A-12C show a further embodiment 1200 for supplying particles 902to a detection region. FIG. 12A shows a top view of the currentembodiment 1200 that includes an annotation to indicate the centerlineof the cutaway views in FIGS. 12B and 12C. According to the figures, athird dimension is considered. Here, the top surface of the detectionregion 818 contains a first aperture 1202 that is large enough to passparticles 902 and a second aperture 1204 that is smaller than theparticles. When an electric potential 1206 is applied along thedetection region 818, the fluid and particles 902 will flow out of thefirst aperture 1202 by electroosmosis. The fluid will flow back throughthe second aperture 1204, but the particles 920 will not pass. Thismethod creates local high density of particles 902 at the secondaperture 1204.

According to one aspect the invention is for use in the field of wateranalysis. An exemplary system provides a method and device for on-site,field-based analysis of aqueous samples. Existing water measurementsrequire collecting samples at a variety of locations, returning thosesamples to a laboratory, and processing the samples to findconstituents. The process is time-consuming and expensive, resulting inmany contaminants never being considered.

The system according to the current invention allows measurementson-site, providing analysis of hundreds or thousands of analytes in onetest. A technician collects a water sample, processes that sample foranalysis, and then introduces the sample to the cartridge. The cartridgeis inserted into a housing, and appropriate analysis options are chosenthrough the user interface. Optics and electronics within the housingprocess the sample, analyze and measure the water constituents, andprovide specific, quantitative feedback to the technician regarding allwater contaminants.

The use of Raman spectroscopy, for example, provides a specificfingerprint for a wide variety of compounds. Coupling with additionaloptical and electrical measurement techniques allows better, faster, andmore accurate analysis. The data resulting from the analysis providesinformation on a large number of analytes, eliminating the need fortedious, repetitive, expensive laboratory processing.

In some cases, users are interested in measuring one or morecontaminants. For example, oil refineries are closely regulated forcertain heavy metals, such as selenium, mercury, and lead. Agriculturalusers need to monitor the water applied to crops to avoid microorganismcontamination. In one aspect of the invention, the cartridge and housingare modified for these specific measurements through selection ofdetection particles and optical measurement techniques. Thesemodifications speed analysis and improve sensitivity.

In another aspect of the invention an option for food analysis isprovided. Concerns continue to grow over contamination in our foodsupply. Food received from overseas sources might not meet requiredstandards; fruits and vegetables can be contaminated with microorganismssuch as E. coli; consumers may have allergies to specific foods. Thehousing and cartridge are modified to measure and report on these foodcontaminants. The user dissolves a food sample in a buffer solutionbefore analysis. Instead of providing specific quantitative feedback,the system provides a “go/no-go” result indicating the presence of acontaminant of concern.

FIG. 13A shows a front view of the analysis instrument 1300,illustrating a screen 1302 to present information to the user andbuttons 1304 for user input. FIG. 13B shows a bottom view of theinstrument 1300, illustrating at least one cartridge input slot 1306 forinserting the analysis cartridge (see FIGS. 17 ).

FIG. 14 shows an example optical system 1400, contained within theanalysis instrument 1300. Shown is a light source 1402, such as a laser,projecting a light beam 1406 passing through a series of optics 1408arranged as a beam expander that is reflected into a dichroic optic 1410to direct the reference light beam 1411 into a spectrometer 1412 foranalysis in a monochrometer 1414 and recordation in a CCD array 1416.The dichroic 1410 simultaneously directs the signal light beam 1413 tothe cartridge 1418 to gather a signal from a sample in the cartridge1418 and reflect the signal along the beam path into the spectrometer1412 and CCD 1416 array for analysis.

FIG. 15A shows a block diagram presenting the system subcomponents 1500.The instrument provides a user input 1502 for inputting samples, controlagents, nanoparticles, etc. and selecting analysis options. The systemfurther includes a user output 1504 for outputting the samples, controlagents, nanoparticles, etc. and displaying, printing or outputtingsystem results. Control electronics 1506 monitor system integrity andperformance, operate the electric field applied to the cartridge 1508,and operate the optical system 1510 that optically reads data from thecartridge 1508 according to the optical system 1510 as described above.The control electronics 1506 digitize and process the optical data,presenting the results to the user on an external interface 1512. Thecontrol electronics 1506 and external interface 1512 further provideconnectivity with external devices for data transfer, through variousmeans such as wireless or wired connections.

FIG. 15B. A flowchart illustrating system use that includes placing thesample in the cartridge 1520, loading the cartridge in the instrument1522, selecting the analysis options and starts the analysis 1526 by thesystem 1500. The analysis options can include expected contaminants,measurement accuracy, or analysis time, plus sample information for datatracking, such as location, temperature, quantity, user, or other testidentification information. When the system 1500 is started, acalibration standard is injected 1528, then the sample is injected 1530and the separation flow is started 1532 and optical data and other dateis collected 1534, where separation flow 1532 and data collection 1534iteratively continue 1536 until completion. The data is analyzed 1538and the results are presented 1540.

FIG. 16 shows exemplary data 1600 presented to the user.

FIGS. 17A-17B. show top and side views of the analysis cartridge 1700,respectively. FIG. 17A shows a top view having at least one sample inlet1702 and at least one sample outlet 1704, in addition to at least onebuffer inlet 1706 and at least one buffer outlet 1708. Further shown isat least one detection material inlet 1710, at least one detectionmaterial outlet 1712, at least one calibration standard inlet 1714, atleast one calibration standard outlet 1716 and a detection window 1718disposed above the detection region of the channel, as described above,where the analysis cartridge is surrounded by a protective shell 1720.FIG. 17B shows a side view of the analysis cartridge 1700 where theanalysis chip is surrounded by the protective shell 1720, that holdschip base 1722, a chip lid 1724, a sealing gasket 1726, and protectivefilm cover 1728.

According to another embodiment of the invention, the use of calibrationstandards is an important procedure for chemical separations andchemical analysis. Calibration standards provide a reference pointagainst which data may be compared to provide accurate quantitativeresults.

For example, in chromatographic separations, chemicals with knownretention times may be added to the solution under analysis. As thesechemical elute from the chromatographic column, their elution timeprovides a scale against which the elution time of unknown species maybe compared. Effectively, calibration standards provide a ruler forcalibrating the elution time.

Calibration standards are also useful as a ruler for signal intensity.When a known amount of calibration standard is introduced to a sample,the quantity of unknown materials may be determined by comparing themeasurement intensity, often through ratiometric methods.

The current embodiment of the invention provides calibration standardsfor chemical analysis using Raman spectroscopy and hand-heldmicrofluidic testing devices. The introduction of calibration standardsat device manufacturing or during analysis creates a powerful,quantitative chemical analysis system. For example, one analyticalmethod that has been greatly hindered by a lack of reproducibility andaccuracy is surface-enhanced Raman spectroscopy. Surface-enhanced Ramanspectroscopy relies upon nanoscale metallic nanoparticles (i.e.,markers) to provide an amplified Raman response. The current embodimentof the invention provides for calibration standards that greatly improvethe reproducibility and accuracy of these surface-enhancingnanoparticles.

FIG. 18 shows a hand-held microfluidic testing device, according to oneembodiment, that includes a housing having a cartridge receiving port, acartridge for input to the cartridge receiving port having a sampleinput and a channel, where the channel includes a mixture ofRaman-scattering nanoparticles and a calibration solution, where thecalibration solution includes chemical compounds capable of interactingwith a sample under test input to the cartridge and the Raman-scatteringnanoparticles, and an optical detection system in the housing, where theoptical detection system is capable of providing an illuminated electricfield, where the illuminating electric field is capable of being usedfor Raman spectroscopy with the Raman-scattering nanoparticles and thecalibration solution to analyze the sample under test input to thecartridge.

In one embodiment of our invention, the calibration standard is anisotope of the analyte under investigation. An isotope is a powerfulinternal standard as it differs from the analyte only in the number ofneutrons. The chemical response and reaction will be nearly identical tothe analyte. However, under many forms of spectroscopy, including Ramanspectroscopy, the isotope has a different spectrum. Therefore, one canmeasure the analyte and isotope spectra simultaneously, usingratiometric analysis to quantify the unknown analyte.

An alternative embodiment relies upon an isotopic calibration standardfor a compound that is chemically similar to the analyte under study.For example, selenium and sulfur are chemically quite similar. Ameasurement of selenate may rely upon the measurement of sulfate, of aselenate isotope, or of a sulfate isotope as a calibration standard.

Another embodiment of our invention provides a calibration standardduring manufacturing through modification of nanoparticle markerchemistry. Modifications of the marker provide two advantages. First,the modification can be chosen for analyte specificity using compoundsdesigned to interact only with the analyte under investigation. Thisapproach reduces interferences while increasing signal strength. Second,the modification provides a well-defined Raman signal that measures thesignal enhancing capabilities of the markers. This signal acts as acalibration standard.

In one aspect of the invention, the chemical compounds can includethiols, amines, silanes, polymeric particles, metallic particles, crownesters, cysteamine, cystamine, diethylaminethanethiol, mercaptopropionicacid, 1-propanethiol, octanethiol, octyldecanethiol, polystyrene, iron,or silica.

According to another aspect of the invention, the calibration solutioncomprises isotopes of the sample under test.

In a further aspect of the invention, the calibration solution comprisesa chemical composition in the sample under test.

The present invention has now been described in accordance with severalexemplary embodiments, which are intended to be illustrative in allaspects, rather than restrictive. Thus, the present invention is capableof many variations in detailed implementation, which may be derived fromthe description contained herein by a person of ordinary skill in theart. For example, the device may be injection molded, constructed ofelastomers, or processed using semiconductor methods and materials. Thechannels may contain curved segments to extend their lengths or may havevarying depths to encourage separation. The detection particles couldcombine multiple signaling and binding mechanisms, such as beingmagnetic and fluorescent to enhance optical detection within a magneticfield. Example channel shapes and sizes (heights, ratios), materials,electrode configurations, carrier solutions, fabrication methods can bevaried without departing from the spirit of the invention. All suchvariations are considered to be within the scope and spirit of thepresent invention as defined by the following claims and their legalequivalents.

We claim:
 1. A portable testing device comprising: a. a housing, whereinsaid housing comprises a cartridge receiving port; b. a cartridge forinput to said cartridge receiving port, wherein said cartridge containsa mixture including a water sample comprising an analyte,Raman-scattering nanoparticles and a calibration solution, wherein saidcalibration solution comprises a chemical compound capable ofinteracting with the analyte and the Raman-scattering nanoparticles; andc. an optical detection system in said housing, wherein said opticaldetection system is capable of providing an illuminated electric field,wherein said illuminating electric field is capable of being used forRaman spectroscopy with said Raman-scattering nanoparticles and saidcalibration solution to analyze said sample under test input to saidcartridge, wherein the chemical compound is selected from the groupconsisting of thiols, amines, silanes, polymeric particles, metallicparticles, crown esters, cysteamine, cystamine, diethylaminethanethiol,mercaptopropionic acid, 1-propanethiol, octanethiol, octyldecanethiol,polystyrene, iron, and silica.
 2. The portable testing device in claim 1wherein, said calibration solution further comprises an isotope of theanalyte.
 3. The portable testing device in claim 1 wherein the analyteis selenate and said calibration solution further comprises a selenateisotope or a sulfate isotope.
 4. A portable testing device comprising:a. a housing, wherein said housing comprises a cartridge receiving port;b. a cartridge for input to said cartridge receiving port, wherein saidcartridge contains a mixture including a water sample comprising ananalyte, Raman-scattering nanoparticles and a calibration solution,wherein said calibration solution comprises a chemical compound; and c.an optical detection system in said housing, wherein said opticaldetection system is capable of providing an illuminated electric field,wherein said illuminating electric field is capable of being used forRaman spectroscopy with said Raman-scattering nanoparticles and saidcalibration solution to analyze said sample under test input to saidcartridge, wherein said chemical compound is selected from the groupconsisting of thiols, silanes, crown esters, cysteamine, cystamine,diethylaminethanethiol, mercaptopropionic acid, 1-propanethiol,octanethiol, octyldecanethiol and polystyrene.
 5. The portable testingdevice in claim 4 wherein, said calibration solution comprises anisotope of the analyte.
 6. The portable testing device of claim 4wherein said chemical compound is selected from the group consisting ofthiols, diethylaminethanethiol, cysteamine and cystamine.